The aim of this study is to develop the clinical potential of multitubular biological reactors for removal of methotrexate from serum by establishing optimum design and operating conditions for short-term as well as long-term application with extracorporeal shunts. Novel multitubular hollow fiber reactors with high surface area and low holdup volume will be designed and used for immobilization of carboxypeptidase G1 (CPG1), an enzyme that degrades methotrexate into 2,4-amino, 10N-methylpteroate and thereby renders it harmless. The chemical nature, pore structure and overall dimensions of the capillary membranes will be optimized in order to obtain a suitable support system for stable and highly active enzyme reactors to be used in extracorporeal circulation. Particular attention will be paid to mass transfer from the bloodstream in the lumen to the enzyme sites because the efficiency of such devices is expected to be determined by the rate of substrate diffusion to the reaction zone. The kinetic behaviour of the reactors will be tested both in vitro and in vivo and the experiments will also serve to optimize reactor stability and minimize the untoward hematological effects in dogs after administration of methotrexate. Carboxypeptidase G1 reactors are expected to be useful adjuncts for cancer chemotherapy in methotrexate rescue. Furthermore, they may serve as models for development of similar devices to be used in enzyme therapy.